| 1. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae
- 2021
-
Ingår i: mSystems. - 2379-5077. ; 6:4
-
Tidskriftsartikel (refereegranskat)abstract
- CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions, and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for the industrial conversion of lignocellulosic biomasses. Tolerance to acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of .9,000 strains, where .98% of all essential and respiratory growth-essential genes were targeted with multiple guide RNAs (gRNAs). The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where high-resolution CRISPRi library screening paves the way to understanding and bioengineering the robustness of yeast against acetic acid stress.
|
|
| 2. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2018
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil based biochemical with its renewable counterpart requires the production to be significantly more efficient and price competitive. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulose hydrolysate. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. Numerous attempts have been reported to improve the stress tolerance of Saccharomyces cerevisiae by different bioengineering strategies such as deletion/overexpression of genes. However, the inability to achieve a fine balance of the transcriptional expression of the target and the ancillary gene(s) is one of the major factors that impedes the efficiency of many of these strategies. In this project, we apply CRISPR interference (CRISPRi) technology to investigate the potential of fine-tuning the expression of genes that are related to the stress regulation. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using a sgRNA. Strains with altered regulation will be screened for inhibitor tolerance. Furthermore, transcriptomics analysis of tolerant mutants will be conducted to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate. Here, we will show data on a methodology that we have developed for studying hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates.
|
|
| 3. |
- Torello Pianale, Luca, 1995, et al.
(författare)
-
Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2019
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil-based chemicals with renewable biochemicals requires the production to be significantly more efficient and price competitive. Remediation of several technical bottlenecks is needed before this can be accomplished. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulosic hydrolysates. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. In this project, we are trying to fine tune the expression of stress related genes to boost the stress tolerance in Saccharomyces cerevisiae using the CRISPR interference (CRISPRi) technology. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using an sgRNA. Using a high-throughput yeast transformation method developed in our laboratory, we are generating a CRISPRi strain library. Each strain in this library has altered regulation for at-least one stress related gene. Next, high-throughput phenotypic evaluation of this library is performed by growing the strains under the exposure of inhibitors relevant to lignocellulosic hydrolysates. Here, we will demonstrate our primary CRISPRi library data. Further, we will explain the high-throughput methodologies for generating the CRISPRi mutants and to study their hydrolysate tolerance, adaptation and ethanol production capacity at microscale. In future, we will perform transcriptomics analysis of the most tolerant mutants to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate.
|
|
| 4. |
- Holt, Sylvester, et al.
(författare)
-
Bioflavoring by non-conventional yeasts in sequential beer fermentations
- 2018
-
Ingår i: Food Microbiology. - : Elsevier BV. - 0740-0020 .- 1095-9998. ; 72, s. 55-66
-
Tidskriftsartikel (refereegranskat)abstract
- Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compoundsand their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomycesspecies and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species.
|
|
| 5. |
- Mukherjee, Vaskar, et al.
(författare)
-
CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae
- 2023
-
Ingår i: Engineering Microbiology. - : Elsevier Inc.. - 2667-3703. ; 3:2
-
Tidskriftsartikel (refereegranskat)abstract
- Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates. © 2023 The Author(s)
|
|
| 6. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
High throughput screening of yeast strains for desirable stress tolerant traits for bioethanol production
- 2013
-
Ingår i: Yeast. - : Wiley. - 0749-503X.
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Implementation of very high gravity (VHG) fermentation technology in second generation bioethanol production using raw lignocellulosic biomass is fundamental to establish a commercially viable plant. However, so far the application of this technology is greatly restricted by the unavailability of a fermentative microorganism, resistant enough to the wide variety of stressors commonly encountered in VHG fermentation. In addition, the appropriate tools and knowledge to select such multi-stress tolerant microorganisms and to make a scientifically proven choice of the appropriate candidate strains have been lacking until recently. In this study we screened a large yeast culture collection, consisting of about 700 Saccharomyces cerevisiae and non-Saccharomyces strains from diverse origins, for different desirable traits for bioethanol production. These included, for example, osmotolerance, halotolerance, ethanol tolerance, thermotolerance, and tolerance against fermentation inhibitors like furfural and hydroxymethyl furfural as well as some heavy metals. To this end, a high throughput semi-automated robot was used for spotting up to 96 strains per screening plate. After incubation, plates were scanned and growth was recorded and analyzed using dedicated software. Cluster analysis showed clear differences in tolerance among species and among strains of the same species. In addition, strains showing co-tolerance against different traits could be identified. As such, our study enabled to efficiently select top candidate strains having desirable traits for VHG bioethanol production.
|
|
| 7. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
Phenomics, transcriptomics and metabolomics for identifying concentration-dependent chemical interactions and understanding the mechanistic basis of the mixture toxicity
- 2019
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The prevalence of mixtures of synthetic and natural chemicals in the environment is a growing concern for public health and environmental effects. Currently, most chemical legislations are based on the risk assessments carried out on individual substances and theoretical estimates of combination effect. However, exposure to multi-component mixtures may stimulate unpredicted overall toxic responses due to interactions, where interactions were scored as deviations from the independent action model. In our project, we investigated the frequency and magnitude of interactions in mixtures of five compounds - NaCl, HgCl2, paraquat, rapamycin, clotrimazole - with relatively known specific mode of action. Growth effects by all-combination pair-wise mixtures spanning a wide concentration range were investigated by employing high-resolution yeast phenomics. The baker’s/brewer’s yeast Saccharomyces cerevisiae and the marine yeast Debaryomyces hansenii are used in this study to identify evolutionary conserved mixture effects, with the aim to identify generic responses of relevance to a vast array of organisms. Our results clearly show that both synergistic and antagonistic relationships exist among the tested chemicals and some of these relationships are concentration-dependent. Evolutionary conserved interactions on the level of rate of growth were found for salt and rapamycin (synergy) as well as for salt and paraquat (antagonism). The mechanistic basis of the chemical interactions identified in our study was investigated by transcriptomics and metabolomics. As one example, we observed that several genes with symporter activity and with cation transmembrane transporter activity is downregulated in salt plus paraquat mixtures, while the expression of genes that are related to cofactor-dependent metabolic pathways is stimulated. We believe that the repression of symporter and ion transmembrane transport activity reduces paraquat entry to the yeast cells and thereby reduces its toxic response when combined with salt. On the other hand, upregulation of several of the genes (such as PGI1, PFK1, FBA1, and CDC19) related to cofactor-dependent metabolic pathways boost yeast fermentative activity. Since paraquat induces the production of reactive oxygen species (ROS) via respiration, a shift from aerobic respiration to anaerobic fermentation can reduce formation of ROS, thus reduces oxidative stress by paraquat.
|
|
| 8. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production
- 2014
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:9483
-
Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.
|
|
| 9. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation
- 2017
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. Results: Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multitolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Conclusion: Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.
|
|
| 10. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
-
Polygenic analysis of high osmotolerance in Saccharomyces cerevisiae
- 2015
-
Ingår i: Abstracts of the 27th International Conference on Yeast Genetics and Molecular Biology. - : Wiley. ; 32:S1
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The main objective of our research is to investigate the molecular basis of superior osmotolerance in Saccharomyces cerevisiae and to identify unique mutations in the causative genes that are responsible for superior fermentation performance under very high gravity fermentation. We employed pooled-segregant whole-genome sequence analysis, a technology for efficient polygenic analysis of complex traits developed in our laboratory. For that purpose, a haploid segregant of an osmotolerant yeast strain with the best superior phenotype has been crossed with a haploid segregant from an unrelated industrial strain with a comparatively inferior phenotype. The diploid hybrid has been sporulated and about 30 segregants with the superior phenotype have been selected to construct the superior pool. About 30 segregants were also randomly selected regardless of their phenotype to construct the unselected pool. Pooled genomic DNA extraction was performed for both pools separately and submitted to custom whole-genome sequence analysis. The two parent strains have also been sent for sequencing to determine all SNPs. The variant frequency of the SNPs in the pool has been used to map the QTLs containing the causative mutations in the genome. Several clear QTLs with different strength have been identified in this way. This is followed by the application of reciprocal hemizygosity analysis to identify the causative gene(s) with the responsible mutation in the mapped loci. Finally the identified causative mutations will be introduced in to industrial strains to improve the very high gravity bioethanol fermentation performance.
|
|
| 11. |
- Radecka, Dorota, et al.
(författare)
-
Looking beyond Saccharomyces: the potential of non-conventional yeast species for desirable traits in bioethanol fermentation
- 2015
-
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:6
-
Forskningsöversikt (refereegranskat)abstract
- Saccharomyces cerevisiae has been used for millennia in the production of food and beverages and is by far the most studied yeast species. Currently, it is also the most used microorganism in the production of first-generation bioethanol from sugar or starch crops. Second-generation bioethanol, on the other hand, is produced from lignocellulosic feedstocks that are pretreated and hydrolyzed to obtain monomeric sugars, mainly D-glucose, D-xylose and L-arabinose. Recently, S. cerevisiaerecombinant strains capable of fermenting pentose sugars have been generated. However, the pretreatment of the biomass results in hydrolysates with high osmolarity and high concentrations of inhibitors. These compounds negatively influence the fermentation process. Therefore, robust strains with high stress tolerance are required. Up to now, more than 2000 yeast species have been described and some of these could provide a solution to these limitations because of their high tolerance to the most predominant stress conditions present in a second-generation bioethanol reactor. In this review, we will summarize what is known about the non-conventional yeast species showing unusual tolerance to these stresses, namely Zygosaccharomyces rouxii(osmotolerance), Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha(thermotolerance), Dekkera bruxellensis (ethanol tolerance), Pichia kudriavzevii (furan derivatives tolerance) and Z. bailii (acetic acid tolerance).
|
|
| 12. |
- Ruyters, Stefan, et al.
(författare)
-
Assessing the potential of wild yeasts for bioethanol production
- 2015
-
Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 42:1, s. 39-48
-
Tidskriftsartikel (refereegranskat)abstract
- Bioethanol fermentations expose yeasts to a new, complex and challenging fermentation medium with specific inhibitors and sugar mixtures depending on the type of carbon source. It is, therefore, suggested that the natural diversity of yeasts should be further exploited in order to find yeasts with good ethanol yield in stressed fermentation media. In this study, we screened more than 50 yeast isolates of which we selected five isolates with promising features. The species Candida bombi, Wickerhamomyces anomalus and Torulaspora delbrueckii showed better osmo- and hydroxymethylfurfural tolerance than Saccharomyces cerevisiae. However, S. cerevisiae isolates had the highest ethanol yield in fermentation experiments mimicking high gravity fermentations (25 % glucose) and artificial lignocellulose hydrolysates (with a myriad of inhibitors). Interestingly, among two tested S. cerevisiaestrains, a wild strain isolated from an oak tree performed better than Ethanol Red, a S. cerevisiae strain which is currently commonly used in industrial bioethanol fermentations. Additionally, a W. anomalus strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time. Other non-Saccharomycesyeasts yielded lower ethanol amounts.
|
|
